JP2013184921A - Expression modulator for clock gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a substance that can be used in a broad range of applications and effectively regulate clock gene expression.SOLUTION: A clock gene expression regulating agent comprises as an active ingredient, one or more selected from the group consisting of caryophyllene oxide and compounds represented by structural formula (wherein X is CH, NHor NHCH).

Description

  The present invention relates to a clock gene expression regulator and a circadian rhythm regulator comprising the same.

  Almost all living organisms on the earth have “biological clocks” that vibrate autonomously in a cycle of about 24 hours. Biological clocks cause biological diurnal fluctuations called circadian rhythms, including the sleep / wake cycle of organisms, body temperature, blood pressure, hormone secretion, metabolism, as well as psychosomatic activity, feeding, etc. It is thought that it controls the diurnal variation of various biological phenomena (activities). In recent years, disturbance of circadian rhythm has been pointed out as a cause of various psychosomatic symptoms or diseases such as sleep disorders, skin diseases, lifestyle-related diseases, and neuropsychiatric diseases such as mania.

  As shown in Fig. 1, the biological clock is controlled by a rhythm generation system that includes a group of genes called "clock genes". In the case of mammals, the four cores CLOCK, BMAL1, PERIOD, and CRYPTOCHROME The feedback loop formed by promoting / suppressing the transcription of the gene encoding the protein forms the core of the clock molecular mechanism, and this feedback loop engraves a rhythm of about 24 hours, thereby forming a circadian rhythm. The

  Clock genes directly control the expression rhythm of other genes as transcription factors, and indirectly control the daily expression of more genes through regulation of hormone secretion, etc. It has been clarified that when the rhythm is disturbed, the function of the living organs and the endocrine system are impaired, causing various diseases including lifestyle-related diseases such as hypertension (Non-patent Document 1). For example, obese people have been reported to have an abnormality in the expression of clock genes, and have been reported to be associated with depression and cancer. Furthermore, it has been found that the clock gene regulates circadian rhythms of various physiological functions of the skin, and in experiments using normal human skin fibroblasts, the type I collagen gene is similar to the clock gene Period2. It is reported that the expression pattern is expressed with circadian rhythm (Non-patent Document 2).

  The circadian rhythm control center (central clock) is located in the suprachiasmatic nucleus of the hypothalamus, but clock genes are also expressed in peripheral tissues such as the liver, kidney, and skin, and the circadian rhythm is formed by a similar system. It has become clear to do. Peripheral clock genes are regulated by signals from the suprachiasmatic nucleus, but signal stimulating factors such as glucocorticoids, catecholamines, and angiotensin II directly regulate the expression of clock genes in peripheral tissues and cells. It has also been found that it controls and creates physiological rhythms. Recently, in cultured cells such as dermal fibroblasts, stimulating factors such as glucocorticoid, forskolin, or serum are added to synchronize the expression rhythm to induce the circadian rhythm of clock gene expression in vitro, and its expression is an indicator The circadian rhythm is evaluated (Non-patent Documents 3-5).

  Furthermore, in such an evaluation system using human skin fibroblasts, herbal medicines such as hinoki extract and chlorella extract and essential oils such as juniper oil and lavender oil regulate the expression of Bmal clock gene, and arnica extract and konpe extract It has been reported that herbal medicines such as Juniper oil and Seder oil can regulate the expression of the Period clock gene (Patent Documents 1 and 2).

  As described above, by adjusting the expression of clock genes, it is possible to adjust various behavioral rhythms and circadian rhythms of physiological functions that are controlled by it, and it can be used for a wide range of applications and is effective. In particular, the development of new drugs that can regulate the expression of clock genes is still strongly desired.

International Publication No. 2011-122040 Pamphlet International Publication No. 2011/122041 Pamphlet

Hastings M., O’Neill JS., And Maywood ES., (2007) Circadian clocks: regulators of endocrine and metabolic rhythms. Journal of Endocrinology, 195; 187-198 Izumi Katsuyo et al., Search for skin physiology genes with circadian rhythm, The 32nd Annual Meeting of the Molecular Biology Society of Japan Abstract 2P-0009 Okamura H., (2004) Clock genes in cell clocks: Roles, Actions, and Mysteries. Journal of Biological Rhythms, 19 (5); 388-399 Balsalobre A., Damiola F., and Schibler U., (1998) A serum shock induces circadian gene expression in mammalian tissue culture cells.Cell, 93; 929-937 Yagita K., Tamanini F., van der Horst G., and Okamura H., (2001) Molecular mechanisms of the biological clock in cultures fibroblasts.Science, 292; 278-281

  In view of the circumstances as described above, an object of the present invention is to provide a novel drug capable of regulating the expression of a clock gene.

  The present inventor has found that caryophyllene oxide and methyl benzoate (methylbenzoate) derivatives can promote the expression of clock genes such as Bmal and Period in cultured cells, and have completed the present invention.

The clock gene expression regulator of the present invention contains at least one selected from the group consisting of caryophyllene oxide and a compound having the following structural formula as an active ingredient.
(X is CH ₃ , NH ₂ or NHCH ₃ )
Until now it has never been known that these specific compounds can regulate the expression of clock genes.

  Although the clock gene expression regulator of the present invention is not limited, for example, Bmal gene (Bmal1, Bmal2), Period gene (Period1, Period2, Period3), Clock gene, Cryptochrome gene, albumin site D-binding protein (Dbp) It regulates the expression of genes, E4BP4 gene, Npas2 gene, Rev-erb gene, among them Bmal, Period, Clock and / or Cryptochrome genes, particularly Bmal and Period genes, which are core genes of biological clocks.

  In the present invention, gene expression regulation includes regulating gene expression rhythm (phase or period) in addition to promoting gene expression.

  The circadian rhythm adjusting agent of the present invention includes the above clock gene expression regulating agent. As mentioned above, the clock genes directly or indirectly control the diurnal expression of various genes involved in the functions of the living organs and the endocrine system, such as Bmal and Period, which are the core genes of biological clocks. By regulating the expression of the clock gene, it is possible to adjust various behavioral rhythms of the living body and physiological circadian rhythms controlled thereby.

  The clock gene expression regulator of the present invention can be used in various forms such as pharmaceuticals, quasi-drugs, cosmetics, foods, miscellaneous goods, clothing, etc. It is possible to ameliorate various psychosomatic symptoms or diseases caused by.

Schematic showing the core loop part of the circadian rhythm generation system using clock genes Graph showing the induction of circadian rhythm of clock gene expression by cortisol in cultured human skin fibroblasts Graph showing the regulation of clock gene Bmal1 expression by test substances in cultured human skin fibroblasts Graph showing the regulation of clock gene Per1 expression by test substances in cultured human skin fibroblasts

  The clock gene expression regulator of the present invention contains caryophyllene oxide or the above-described methyl benzoate (methylbenzoate) derivative as an active ingredient. These compounds are all known compounds, but will be briefly described below.

  Caryophyllene oxide ((1R, 4R, 6R, 10S) -4,12,12-trimethyl-9-methylene-5-oxatricyclo [8.2.0.04,6] dodecane) is a colorless liquid or colorless having a molecular weight of 220.35 It is obtained by epoxidizing caryophyllene or isocalyophylene with crystals in the presence of sodium acetate with peracetic acid at 0-5 degrees. Naturally, it is known to exist in copaiba oil, glove oil, lavender oil and the like.

  Further, methyl 2-methylbenzoate, which is a methyl benzoate derivative used in the present invention, is a colorless liquid having a molecular weight of 150.18. Methyl 2-aminobenzoate is a liquid having a molecular weight of 151.17. Add 80 parts of anthranilic acid to 120 parts of methanol, gradually add 100 parts of concentrated sulfuric acid at 40 degrees or less, and complete the reaction at 75 to 83 degrees. After recovery, it is obtained by adding to a solution of 110 parts of sodium carbonate and 240 parts of water, neutralizing, washing with water and then purifying by distillation. There is also a synthesis method from O-nitrotoluene. Naturally, it is known to exist in neroli, ylang ylang flower essential oil, and the like. Methyl 2-methylaminobenzoate is a colorless liquid having a molecular weight of 165.2 and is known to exist naturally in orange peel, mandarin oil, and the like.

  The clock gene expression regulator of the present invention contains one or more of the above compounds. In the present invention, the compound may be naturally derived or synthesized.

  The clock gene expression regulator of the present invention may be used alone, or may be used in combination with a drug having an action of regulating the expression of other clock genes.

  Furthermore, although the clock gene expression regulator and circadian rhythm regulator of the present invention may be used alone, they can be included in various objects. Depending on the type of the object, optional components can be further included in addition to the essential components.

  For example, when the object is an external preparation for skin, its dosage form (for example, liquid, powder, granule, aerosol, solid, gel, patch, suppository, etc.) or product form (for example, Depending on cosmetics, pharmaceuticals, quasi drugs, etc., any component usually contained in such an external preparation for skin may be included together with the above-mentioned clock gene expression regulator. The topical skin preparation is a concept that encompasses all compositions applied to the skin (including the scalp, hair, and nails). For example, basic cosmetics, makeup cosmetics, hair cosmetics, skin or hair cleansing agents, etc. Cosmetics, ointments, patches, suppositories, various drugs such as dentifrices and quasi drugs. The dosage form is also not particularly limited. For example, an aqueous solution system, a solubilization system, an emulsification system, an oil liquid system, a gel system, a paste system, an ointment system, an aerosol system, a water-oil two-layer system, a water-oil-powder 3 Includes any dosage form, such as a layer. When the skin external preparation is a cosmetic, for example, perfume, eau de toilette, eau de cologne, cream, milky lotion, lotion, foundations, powdered white powder, lipstick, soap, shampoo / rinse, body shampoo, body rinse, body powder And bath agents.

  Also, for example, any miscellaneous goods such as fragrance, deodorant, aroma candle, incense, stationery, wallet, bag, shoes, etc., and any clothing such as underwear, clothes, hats, stockings, socks, etc. The clock gene expression regulator of the present invention can be included in any form of supplements (nutritional supplements) such as powders, granules, tablets, capsules, confectionery, beverages, etc. It may be used in inhalation products such as sprays.

  In addition, although the usage aspect of the clock gene expression regulator of this invention was illustrated, it is not limited to these, As long as the effect of this invention can be achieved, it can be used in arbitrary aspects. In addition to the clock gene expression regulating agent of the present invention, other drugs having a circadian rhythm adjusting action can be added according to a specific embodiment as long as the effects of the present invention are not impaired.

  The blending amount of the clock gene expression regulator of the present invention in the object can be appropriately selected depending on the type and form of the compound used, the object, etc., and is not particularly limited. For example, for the total mass of the object 0.00001-100 mass%, more preferably 0.0001-50 mass%, and still more preferably 0.0001-20 mass%.

  The specific application of the clock gene expression regulating agent or circadian rhythm adjusting agent of the present invention or an object containing the same is not particularly limited as long as it relates to the circadian rhythm adjustment. For example, jet lag syndrome, shift work syndrome, sleep phase delay syndrome, non-24-hour sleep / wake syndrome, depression with circadian rhythm sleep disorder, and insomnia associated with circadian rhythm disorder, physical condition It can be applied to the prevention, improvement or treatment of various symptoms such as attention deficit, reduced motivation and rough skin.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to the following Example. As cultured cells, various cells such as dermal fibroblasts, epithelial cells, endothelial cells, pigment cells, adipocytes, and nerve cells can be used. In this example, evaluation was performed using human dermal fibroblasts. It was. Since the core system of the clock gene is common to all living species and cell types, it is considered that the evaluation results in human skin fibroblasts can be reflected in other living species and cell types. In addition, the presence of Bmal, Period, Clock, Cryptochrome, albumin site D-binding protein (Dbp), E4BP4, Npas2, Rev-erb, etc. are known as clock genes. The expression of Bmal and Period involved was measured. The presence of Bmal1 and Bmal2 as Bmal and Per1, Per2, and Per3 as Periods are known, but the same gene group is considered to exhibit the same behavior, and they were represented by bmal1 and Per1, respectively. In the following examples, the expression of human Bmal1 (hBmal1) and human Per1 (hPer1) was measured. Moreover, the test substance used below used what purchased the commercial item.

Induction of circadian rhythm of clock gene expression using cultured human skin fibroblasts It was confirmed that circadian rhythm of clock gene expression can be induced in a system using cultured human skin fibroblasts.

As cultured human skin fibroblasts, normal adult skin-derived fibroblasts were purchased (Cell Application, Inc) and used for experiments. The cells were seeded on a DMEM medium supplemented with 10% FBS, 20 mM HEPES, Glutamax, and an antibacterial agent, and cultured at 37 ° C. and 5% CO ₂ . On the sixth day of culture, 50 ng / ml of cortisol was added and treated for 2 hours, and then the cells were sampled over time. RNA was extracted from the cells using a commercially available RNA extraction kit, and the expression levels of hBmal1 and hPer1 genes were measured by RT-PCR using a commercially available PCR primer (Perfect Real Time Primer, Takara Bio Inc.). Similarly, the expression level of the housekeeping gene RPLP0 was quantified and used as an internal standard, and the relative expression level of the target gene relative to the expression level of RPLP0 was calculated.

  In vivo, glucocorticoids such as cortisol are involved in the regulation of biological clocks such as peripheral tissues, and are considered to increase the blood concentration of cortisol when waking up in the morning and reset the biological clock. In cultured cells, individual cells usually divide their rhythms at different times, but by stimulating with a signal stimulating factor such as cortisol, the clock gene expression rhythm can be synchronized to induce circadian rhythms. .

  The results of measuring hBmal1 and hPer1 are shown in FIG. By stimulating with cortisol, it was confirmed that all were expressed with a circadian rhythm with a cycle of about 24 hours.

Evaluation of clock gene expression regulating effect From the above results, it was confirmed that in the cultured human skin fibroblast evaluation system, the clock gene has a circadian rhythm by stimulation with cortisol. Since evaluation in a time zone with a high expression level is desirable, hBmal1 was evaluated for the effect of regulating the clock gene expression of the test substance using the gene expression level 16 hours after stimulation and hPer1 2 hours after stimulation, respectively.

  In the same manner as described above, normal adult skin-derived fibroblasts (Cell Application, Inc) were seeded, and on the 6th day of culture, each drug was added to 50 ppm, and the cells were obtained after 2 hours and 16 hours. Was sampled. As a control, the same amount of ethanol was added instead of the test substance. RNA was extracted from the cells using a commercially available RNA extraction kit, and the expression levels of hBmal1 and hPer1 genes were measured by RT-PCR using commercially available PCR primers (Perfect Real Time Primer, Takara Bio Inc.). Similarly, the expression level of the housekeeping gene RPLP0 was quantified and used as an internal standard, and the relative expression level of the target gene relative to the expression level of RPLP0 was calculated. Dunnett's multiple comparison test was performed on the obtained values, and those having a significant difference in the risk rate of 5% on one side compared with the control were judged to be effective.

  FIG. 3A shows the relative expression level of the hBmal1 gene 16 hours after the addition of the test substance, and FIG. 3B shows the relative expression level of the hPer1 gene 2 hours after the addition of the test substance.

Table 1 below shows the relative expression levels of the hBmal1 gene after 16 hours and the hPer1 gene after 2 hours.

  Caryophyllene oxide, methyl 2-methylbenzoate, methyl 2-methylaminobenzoate, and methyl aminobenzoate (methylanthranilate) significantly increased both the expression levels of hBmal1 and hPer1 genes compared to controls, It has been shown that these compounds can regulate clock gene expression.

(Formulation example)
Hereinafter, although the example of a clock gene expression regulator of this invention is shown, implementation of this invention is not limited to the following. In addition, in the following, all compounding quantities are represented by the mass% with respect to the total product.

Fragrance (1) Alcohol 75.0
(2) Purified water Residual (3) Dipropylene glycol 5.0
(4) Clock gene expression regulator of the present invention: Caryophyllene oxide 10.0
(5) Antioxidant 8.0
(6) Appropriate amount of dye (7) Appropriate amount of UV absorber

Room Fragrance (1) Alcohol 80.0
(2) Purified water Residue (3) Antioxidant 5.0
(4) Clock gene expression regulator of the present invention: methyl 2-methylbenzoate 3.0
(5) 3-Methyl-3methoxybutanol 5.0
(6) Dibenzylidenesorbitol 5.0

Incense (1) Tab powder 75.5
(2) Sodium benzoate 15.5
(3) Clock gene expression regulator of the present invention: methyl 2-methylaminobenzoate 5.0
(4) Eucalyptus oil 1.0
(5) Purified water residue

Bath salt (1) Sodium sulfate 45.0
(2) Sodium bicarbonate 45.0
(3) Lavender oil 9.0
(4) Clock gene expression regulator of the present invention: methyl 2-aminobenzoate 1.0

Massage Gel (1) Erythritol 2.0
(2) Caffeine 5.0
(3) Oat extract 3.0
(4) Glycerin 50.0
(5) Carboxyvinyl polymer 0.4
(6) Polyethylene glycol 400 30.0
(7) edet trisodium 0.1
(8) Polyoxylene (10) Methyl polysiloxane copolymer 2.0
(9) Squalane 1.0
(10) Potassium hydroxide 0.15
(11) Clock gene expression regulator of the present invention: Caryophyllene oxide 0.5
(12) Clock gene expression regulator of the present invention: methyl 2-methylbenzoate 0.5

Massage cream (1) Solid paraffin 5.0
(2) Beeswax 10.0
(3) Vaseline 15.0
(4) Liquid paraffin 41.0
(5) 1.3-butylene glycol 4.0
(6) Glycerol monostearate 2.0
(7) POE (20) sorbitan monolaurate 2.0
(8) Borax 0.2
(9) Caffeine 2.0
(10) Preservative appropriate amount (11) Antioxidant appropriate amount (12) Clock gene expression regulator of the present invention: Caryophyllene oxide 0.5
(13) Clock gene expression regulator of the present invention: methyl 2-methylaminobenzoate 0.5
(14) Purified water residue

Aromatic fiber A microcapsule (particle diameter of 50 μm or less, microparticles containing a clock gene expression regulator of the present invention in a cupro ammonium cellulose solution (cellulose concentration 10% by weight, ammonium concentration 7% by weight, copper concentration 3.6% by weight). (The ratio of the compound in the capsule is 50% by weight) within a range of 0.1 to 20% by weight of the cellulose weight, and after mixing, spinning according to a normal wet spinning method, through a refining process and a drying process Obtained aromatic fiber.

Granule (1) Sucralose 0.1
(2) Clock gene expression regulator of the present invention: methyl 2-aminobenzoate 0.1
(3) Clock gene expression regulator of the present invention: methyl 2-methylbenzoate 0.1
(3) Flavoring 5.0
(4) Excipient (Theolus) 10.0
(5) Maltitol residual

Tablet (chewable type)
(1) Inositol 11.0
(2) Maltitol 21.0
(3) Sucrose 0.5
(4) White coconut extract (DNA Na) 0.1
(5) Yeast extract 0.1
(6) Clock gene expression regulator of the present invention: methyl 2-aminobenzoate 0.1
(7) Clock gene expression regulator of the present invention: methyl 2-methylaminobenzoate 0.1
(7) Flavoring 5.0
(8) Excipient residue

Tablet (1) Lubricant (sucrose fatty acid ester, etc.) 1.0
(2) Gum arabic aqueous solution (5%) 2.0
(3) Acidulant 1.0
(4) Colorant appropriate amount (5) Clock gene expression regulator of the present invention: methyl 2-methylbenzoate 0.1
(5) Clock gene expression regulator of the present invention: methyl 2-methylaminobenzoate 0.1

(6) Carbohydrate (such as powdered sugar or sorbitol)

Candy (1) Sugar 50.0
(2) Minamata 47.95
(3) Organic acid 2.0
(4) Clock gene expression regulator of the present invention: methyl 2-aminobenzoate 0.05

Gum (1) Sugar 43.0
(2) Gum base 30.95
(3) Glucose 10.0
(4) Minamata 16.0
(5) Clock gene expression regulator of the present invention: methyl 2-methylbenzoate 0.05

  The products of these combination examples can adjust the circadian rhythm of the living body by adjusting the expression of the clock gene by the use test in the typical usage mode of each product form.

Claims (3)

A clock gene expression regulator comprising one or more selected from the group consisting of caryophyllene oxide and a compound having the following structural formula as an active ingredient.
(X is CH ₃ , NH ₂ or NHCH ₃ ) The clock gene expression regulator according to claim 1, wherein the clock gene is Bmal or Period. A circadian rhythm adjusting agent comprising the clock gene expression regulating agent according to claim 1 or 2.